Skin external agents and drugs

ABSTRACT

Skin external agent compositions and drugs characterized by containing fumaric acid diester derivatives represented by following general formula (1), wherein R 1  represents C 1-4  alkylene; R 2  and R 3  represent each liner or branched C 1-8  alkyl; and n is an integer of 2 to 5; which provide epidermal barrier function potentiating agents and skin external agent compositions having a high safety, quickly relieving epidermal permeation barrier function failures and having an effect of maintaining the skin in a cosmetologically healthy state; agents to be used together with HB-EFG expression inducing agents accelerating epidermal turnover, amphiregulin expression inhibitors regulating excessive epidermal proliferation, and retinoic acid or its derivative preventing skin chapping caused by retinoic acid or its derivative; and skin aging relieving agents capable of ameliorating depression in the skin metabolism and thinning in association with aging.

FIELD OF THE INVENTION

The present invention relates to skin external agents such as cosmeticsand medicines. Particularly, the present invention relates to anepidermal permeability barrier function-enhancing agent which enhancesthe epidermal permeability barrier function against environmentalinfluences such as sunburn and quickly ameliorates damaged function ofepidermal permeability barrier such as dry skin to maintain the skin ina dermatological and cosmetologically healthy state.

The present invention relates also to an amphiregulin expressioninhibitor and an epidermal hyperproliferation inhibitor which are usefulfor therapy, prevention, and studies on various diseases involvingexcessive amphiregulin expression and dry skin.

Further, the present intention relates to an ameliorating agent for agedskin which prevents metabolic disruption and thinning of the skin due toaging and is effective in treatment of age spots. Still further, thepresent invention relates to an agent that is used together withretinoic acid or a derivative thereof in order to prevent side effects,such as dry skin, caused by retinoic acid or a derivative thereof.

DESCRIPTION OF THE PRIOR ART

All organisms including the human being are influenced by their externalenvironment. However, higher animals such as mammal have an organ whichminimizes the environmental influence on other important organs andsystems for life. The organ is the skin that has, as its most importantfunction, an epidermal permeability barrier function to prevent foreignsubstances from invading into the body and excessive vaporization ofmoisture from the interior of the body.

The epidermal permeability barrier function can temporarily disrupt dueto diseases or exposure to organic solvents, surfactants or UV-rays,which disturbs the skin's internal environments. If this conditioncontinues, such a risk may arise that pathological bacteria and harmfulchemical substances may invade through the skin into the interior of thebody. Accordingly, it is necessary to quickly restore the epidermalpermeability barrier function. In addition, in the state of disruptionin the epidermal permeability barrier function, the epidermis becomesdry and its surface is covered with scales, which leads to acosmetologically undesirable look.

The epidermal permeability barrier function is formed and maintained bykeratinocyte turnover and keratinization near the stratum corneum. Wherethe epidermal permeability barrier function disrupts, hyperproliferationof keratinocytes occurs and continuous hyperproliferation is consideredto deminish normal keratinocyte turnover and keratinization, whichdelays the recovery of the barrier function.

Psoriasis is a disease with unknown causes, and is accompanied withhyperproliferation of keratinocytes and keratinization insufficiency.Drugs such as steroids and Cyclosporin A, immunosuppressive agent, aregenerally administered for its treatment. However, it is pointed outthat these drugs are dangerous for routine or daily application.Therefore, a drug is desired which reduces and normalizes epidermalhyperproliferation more safely. Similarly, a safe and effective externalagent is in demand for dermatological diseases such as atopic dermatitisaccompanied with disruption of the epidermal permeability barrierfunction.

Appearance of the skin is largely influenced by the skin's overallmetabolic ability and its conditions. For example, when the epidermalpermeability barrier function is disrupted, the skin surface becomes dryand is covered with scales, which is an undesirable state. Byhypometabolism with aging, change in skin tone caused by thinning of theepidermis, dullness, age spot, and wrinkles, lead to loss of beauty inappearance.

The appearance of a beautiful skin is maintained by active keratinocyteturnover and normal keratinization near the stratum corneum. When thekeratinocyte turnover is lowered, keratinization may not proceedsmoothly, resulting in dry skin. Lowered turnover due to aging resultsin dullness in the skin or age spots caused by accumulated melanin. Theepidermal turnover is considered to depend on a cellular mitosis rate inthe basal layer, and a decreased mitosis rate due to aging results inthinner epidermis and wrinkles as well.

The keratinocyte turnover is considered to be controlled by theexpression of epidermal growth factors such as amphiregulin,heparin-binding EGF-like growth factor (HB-EGF), and TGF-α. Inparticular, HB-EGF expressing near the basal layer is considered to bethe greatest contributing factor and, therefore, the epidermal turnoveris up-regulated by inducing the HB-EGF expression [Journal ofInvestigative Dermatology (1998), Vol. 111, pp-715 to 721].

Retinoic acid is known as a very effective external agent and is alsoknown to induce HB-EGF expression. On the other hand, retinoic acid hasside effects such as dry skin and temporary darkening, so that, if it isused as a skin topical agent, physicians must monitor the patients verycarefully. Therefore, patients who need routine prevention andtreatments desire the development of safer drugs and a means to preventsuch side effects.

It is known that the expression of amphiregulin does not occur in theskin showing normal turnover, but is restricted in the epidermisexhibiting hyperproliferation or proliferation in culturedkeratinocytes. Consequently, it is believed that the epidermalhyperproliferation can be inhibited specifically and effectively and,accordingly, functions of the skin are normalized by inhibiting theexpression of amphiregulin. The expression of amphiregulin is involvednot only in the hyperproliferation of the skin, but also in the processof development of skin cancer according to a report. However, anysubstance is not yet known which specifically inhibits the expression ofamphiregulin. Therefore, also for the purpose of research progress, thedevelopment of a drug which inhibits the expression of amphiregulin isstrongly desired.

The inventors found that dimethyl fumarate and diethyl fumarate areeffective for ameliorating the epidermal permeability barrier function(Japanese Patent Application Laid-Open No. 12-178116/2000). In addition,it is known that fumaric acid and fumaric acid esters have a sebumsecretion-promoting function (Japanese Patent Application Laid-Open No.4-18017/1992), and fumaric esters are orally administrated for thetreatment of psoriasis (British Journal of Dermatology 1998:138:456–460,1999:141:424–429). However, it is also known that, when they are usedexternally, perilesional skin irritation, mascular papular rashes, andurticarial reactions are caused and there is a risk of causingsensitization and contact urticaria (Dermatology 1994:188:126–130).

DISCLOSURE OF THE INVENTION

The purpose of the present invention is to provide an epidermal barrierfunction-enhancing agent and a composition for skin external applicationwhich are very safe, quickly ameliorate the epidermal permeabilitybarrier function disruption, and have excellent effects on maintainingthe skin in a cosmetologically healthy state; a HB-EGFexpression-inducing agent to accelerate epidermal turnover; anamphiregulin expression inhibitor to deminish epidermalhyperproliferation; an agent to be used together with retinoic acid or aderivative thereof to prevent dry skin caused by retinoic acid or aderivative thereof; and an ameliorating agent for aged skin whichameliorates hypometabolism and thinning of the skin due to aging.

To attain the aforementioned purposes, the inventors earnestly haveinvestigated effects and safety on skin of various kinds of fumaric aciddiester derivatives, and have found that the fumaric acid diesterderivatives expressed by the following general formula (1) are safe toskin(i.e., not showing sensitization) and have an excellent epidermalpermeability barrier-function enhancing function. The inventors havealso found that a skin external agent composition containing thisderivative has excellent effects of ameliorating the epidermalpermeability barrier function; maintaining the skin in adermatologically and cosmetologically healthy state; inducing theexpression of HB-EGF; inhibiting the expression of amphiregulin;preventing skin thinning due to aging; and preventing dry skin caused byretinoic acid.

The present invention provides an epidermal permeability barrierfunction-enhancing agent, an amphiregulin expression inhibitor, anepidermal hyperproliferation inhibitor, a heparin-binding epidermalgrowth factor-like growth factor (HB-EGF) expression inducing agent, anameliorating agent for aged skin, an agent to be used together withretinoic acid or a derivative thereof, an agent to prevent dry skincaused by retinoic acid, and a skin external agent composition, whichall comprises fumaric acid diester derivatives expressed by thefollowing general formula (1)

wherein R¹ represents a C₁₋₄ alkylene group; R² and R³ each represents alinear or branched C₁₋₈ alkyl group; and n is an integer of 2 to 5.The present invention also provides a skin external agent compositioncomprising the aforesaid fumaric acid diester derivatives, and retinoicacid or a derivative thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments of the present invention will be described below indetail.

Examples of the fumaric acid diester derivatives expressed by theaforementioned general formula (1) used in the present invention includebis(diethylene glycol monoethyl ether) fumarate expressed by thefollowing general formula (2):

such as bis(triethylene glycol monoethyl ether) fumarate, bis(diethyleneglycol monomethyl ether) fumarate, bis(triethylene glycol monomethylether) fumarate, bis(diethylene glycol monobutyl ether) fumarate, andbis(dipropylene glycol monoethyl ether) fumarate. These substances areall known. Those with n=2 or 3 are particularly desirable from theviewpoint of lipophilic/hydrophilic balance.

The fumaric acid diester derivatives expressed by the aforementionedgeneral formula (1) used in the present invention can be manufactured bya known method described in, e.g., Japanese Patent Application Laid-OpenNo. 9-255622/1987.

In the aforementioned general formula (1), to represent the fumaric aciddiester derivatives used in the present invention, the C₁₋₄ alkylenegroups, R¹, can be identical with or different from each other. Whendifferent kinds of the alkylene groups, such as C₂H₄ and C₃H₈, arepresent together, they may be bound randomly or in block-wise. The C₁₋₈linear or branched alkyl groups for R² and R³ can be identical with ordifferent from each other.

A preferable amount of the fumaric acid diester derivative to be blendedin the present skin external agent composition ranges from 0.001 to 15.0mass % (hereinafter simply expressed by “%”) and more preferably from0.1 to 10.0% relative to a total amount of the skin external agentcomposition. If the amount is less than 0.001%, the intended effects ofthe present invention may not be attained sufficiently. On the otherhand, if the amount exceeds 15.0%, there may be no proportional increasein the effects, which may not be desirable sometimes.

Conventional external bases and other pharmaceutical active componentscan be added to the present epidermal permeability barrierfunction-enhancing agent, the amphiregulin expression inhibitor, theepidermal hyperproliferation inhibitor, the heparin-binding epidermalgrowth factor-like growth factor (HB-EGF) expression-inducing agent, theaged skin ameliorating agent, the agent to be used together withretinoic acid or a derivative thereof, and the agent to prevent dry skincaused by retinoic acid or a derivative thereof. The external basesinclude oily bases, water-in-oil and oil-in-water emulsion type bases,and water-based ones. Nonrestrictive examples of the oily bases arevegetable oils, animal oils, synthetic oils, fatty acids, and natural orsynthetic glycerides. As the pharmaceutical components, any ones can beused appropriately as desired, for example, analgetic andanti-inflammatory agents, bactericidal sterilization agents,astringents, skin emollients, hormones and vitamins. In addition, thefollowing compounds can be blended in any combinations: humectants,UV-ray absorbents, alcohols, chelating agents, pH modifiers,antiseptics, thickening agents, pigments, perfumes and plant extracts.

The fumaric acid diester derivatives expressed by the general formula(1) are soluble in water, so that they are advantageously formulated.

The agent to be used together with retinoic acid or a derivative thereofin the present invention can be administrated externally, orally or byinjection when retinoic acid or its derivative is administrated.External administration is desirable. In a skin external agentcomposition as one of the forms of administration, the fumaric aciddiester derivatives expressed by the aforementioned general formula (1)can be formulated together with retinoic acid or a derivative thereof.

A retinoic acid derivative having a physiological activity equivalent tothat of retinoic acid can be used besides retinoic acid.

When retinoic acid or derivative thereof is used together with thefumaric acid diester derivatives expressed by the aforementioned generalformula (1), an amount of retinoic acid or derivative thereof to be usedis not particularly limited, but a preferable amount ranges from 0.001to 1 mass % and more preferably from 0.01 to 0.5 mass %.

Moreover, the aforesaid agents or drugs can be incorporated in usualcosmetics and bath agents as well as in skincare cosmetics. They can bein any dosage form such as lotions, milky lotions, creams, packs, powderand granules. They can be applied to any part of the human skin,including scalp. When applied on the scalp, anti-dandruff and hair toniceffects are expected.

The skin external agent compositions as described above includemedicines for diseases such as psoriasis and atopic dermatitis,quasi-drugs and cosmetics. The cosmetics include general cosmetics suchas skincare cosmetics, makeup cosmetics, cosmetic bases and hair carecosmetics as well as bath agents, among which the skincare cosmetics aremost desirable since they take the full advantage of the presentinvention. They can be in any dosage form such as lotions, milkylotions, creams, packs, powder, granules, gels and ointments.

In addition to the substances mentioned above, vehicles, surfactants andantioxidants allowed to be used in medicines, quasi-drugs or cosmeticscan be blended in the present drugs or the skin external agentcompositions in such an amount that the purpose of the present inventionis achieved.

EXAMPLES

The present invention will be explained with reference to the followingTests, Examples and Comparative Examples.

Test 1 (Epidermal Permeability Barrier Function Amelioration Test)

An effect of improving epidermal permeability barrier function byapplying the agents on the skin of which epidermal permeability barrierfunction has been disrupted was evaluated by the following test method.

1. Experimental Animals Used

Male hairless mice (Hos: Hr-1) of 10 weeks old at the beginning of thetest were used with each 5 mice per group.

2. Measurement of the Epidermal Permeability Barrier State

2-1. Measurement Device and Conditions

Transepidermal water loss (TEWL) was measured as follows using acontinuous perspiration measurement device, Hydrograph AMU-100 (ex K & SCo., Ltd.). A 1 cm² capsule was closely attached to the skin andnitrogen gas was led into the capsule in a rate of 300 mL/min. Theamount of water vapor in the nitrogen gas was measured before the gaswas fed to the capsule and after it was recovered from the capsule. Froma difference between the amounts, the amount of water in mg_vaporizedfrom 1 cm² area of the skin per minute, TEWL, was calculated.

2-2. Samples and the Experimental Method

The samples were prepared by mixing the compounds indicated in Table 1below, e.g., bis(diethylene glycol monoethyl ether) fumarate, with a 50%aqueous ethanol solution (base). The TEWL of the hairless mouse dorsalskin was first measured at the beginning of the test. Subsequently, 0.05mL of each sample was applied on an area of approximately 2.5 cmdiameter in the dorsal skin of the hairless mice once a day from Mondayto Friday (pre-application). Then, on the third day from the last day ofthe pre-application, the skin was irradiated once with ultraviolet B ray(UVB) at a dose of 0.15 J/cm². Each sample was applied immediately afterthe UVB irradiation until the 3^(rd) day from the irradiation in thesame amount and at the same frequency as in the pre-application. TheTEWL was measured on the 3^(rd) and 4^(th) days after the irradiation.TEWL change rates (=TEWL value on the 3^(rd) or 4^(th) day after theirradiation/TEWL value at the beginning of the test) were calculated,which show changes in TEWL relative to the initial TEWL due to theexposure to UVB, and the mean values thereof were compared between thecontrol group and each sample group.

2-3. A Method of Assessment of the Effects

TEWL changes with the degree of disruption of the epidermal permeabilitybarrier function. A higher value of TEWL change rate implies a greaterdegree of disruption of the epidermal permeability barrier function. Inthe case of the application of the base alone, disruption in theepidermal permeability barrier function by UVB was similar to that inthe case of non-application. Therefore, a group showing a lower TEWLchange rate than that of the control group, where the base alone wasapplied and thus showed the natural state of the disruption in theepidermal permeability barrier function, was assessed as available inthe epidermal permeability barrier function amelioration.

The TEWL change rates in the hairless mice after the application of thesamples are as shown in Table 1.

TABLE 1 TEWL change rate Compounds Concentrations, % 3^(rd) day 4^(th)day Bis (diethylene glycol 1 3.25 2.49 monoethyl ether) fumarate — 07.98 7.27 Diphenylethyl fumarate .1I 7.82 6.57 Dimethyl fumarate 1 3.242.50 Mean values

As is seen from the results of this test, bis(diethylene glycolmonoethyl ether) fumarate which is a fumaric acid diester derivativeaccording to the present invention clearly reduces disruption in theepidermal permeability barrier function by UVB and ameliorates epidermalpermeability barrier function, compared to the base alone anddiphenylethyl fumarate, and its epidermal permeability barrier functionamelioration effects is as great as that of dimethyl fumarate which hassensitizing potential. Next, a similar test was carried out usingbis(diethylene glycol monoethyl ether) fumarate in variousconcentrations as described in Table 2 below.

TABLE 2 TEWL change rate Compounds Concentrations, % 3^(rd) day 3^(rd)day Bis (diethylene glycol 0.001 7.01 7.31 monoethyl ether) fumarate Bis(diethylene glycol 0.01 6.46 6.84 monoethyl ether) fumarate Bis(diethylene glycol 0.1 3.79 5.21 monoethyl ether) fumarate Bis(diethylene glycol 1 3.62 4.33 monoethyl ether) fumarate — 0 8.56 8.11Mean valuesTest 2 [Safety (Sensitization) Test]

Safety (sensitization) was evaluated by a maximization test. An area of4×6 cm² of skin on the scapula of a Hartley strain guinea pig (female)weighing 350 to 400 g was shaved and given intradermal injections witheach three injections in two lines according to the followingprocedures:

-   (1)each 0.05 mL of Freunds' Complete Adjuvant (hereinafter simply    called FCA) was intradermally injected at each one spot in the right    and left sites.-   (2) each 0.05 mL of a 3% fumaric acid diester derivative solution in    ethanol of as a test sample was intradermally injected at another    each one spot in the right and left sites.-   (3) to a 6% fumaric acid diester derivative solution in FCA as a    test sample, the same amount of sterilized water was added and    resultant emulsified solution was intradermally injected at a right    side and a left side sites in an amount of 0.05 mL.

At one week after the aforementioned procedures, the skin of the samesites was shaved and 10% of sodium lauryl sulfate in vaseline wasapplied to cause light inflammation. After 24 hours from theapplication, 0.2 mL of a 3% fumaric diester derivative solution inethanol as a test sample, which was applied on a piece of gauze, waspatched close on the same sites for 48 hours. On the 21^(st) day afterthe intradermal injection, the abdominal area was shaved, to which asolution of a fumaric acid diester derivative in ethanolas a testsample, which was applied in a piece of gauze, was patched close for 24hours. The symptoms in the skin were visually examined according to thefollowing criteria after 48 hours.

Symptoms Rating No reaction 0 Scattered mild redness 1 Moderate anddiffuse redness 2 intense redness and swelling 32. Test Results

The sensitization test results with various fumaric acid diesterderivatives after 48 hours are shown below. As seen from the testresults, bis(diethylene glycol monoethyl ether) fumarate, which is afumaric acid diester of the present invention, was confirmed to have nosensitizing potential. However, bis(ethylene glycol monomethyl ether)fumarate which has the general formula (1) with n=1 was found to havesensitizating potential as diethyl fumarate did.

Test samples Positivity Diethyl fumarate 90% Bis (ethylene glycolmonomethyl ether) fumarate 90% Bis (diethylene glycol monoethyl ether)fumarate  0%Test 3 (Human Dry Skin Amelioration Test)

A skin lotion having the composition described below was prepared by theformulation method described below. The sample lotion was applied onskin at an inside of an upper arm of 10 healthy subjects (males of 25 to52 years old) and tested for human dry skin amelioration (epidermalpermeability barrier function recovery test). As a control, one havingthe same composition except for bis(diethylene glycol monoethyl ether)fumarate was used.

Skin lotion composition Amount, % Component (A) Olive oil 10.0 Isopropylmyristate 1.0 Polyoxyethylene (15) nonylphenyl ether 0.5 Propyleneglycol 1.0 Glycerin 2.0 Component (B) Methylparaben 0.1 Ethanol 7.0 Bis(diethylene glycol monoethyl ether) fumarate 1.0 Purified water BalancePreparation Method

Components (A) and (B) were separately dissolved uniformly, and thenwere combined to disperse with stirring. The mixture was filled in acontainer. The contents were shaken to effect uniform dispersion beforeeach use.

Each sample was applied to the test site (2 cm×2 cm for each sample) inan amount of 0.1 mL once a day after bathing, which was continued for 2months (61 days). On the 62^(nd) day from the beginning of theapplication, the skin surface was wiped with acetone to attain 0.15mg/cm²/min of TEWL, indicating an induced state of dry skin.Subsequently, the sample was applied again in the same way as describedabove. Along with investigating how much the epidermal permeabilitybarrier function recovers, and also the skin surface status and thepresence of side effects such as rashes and itching were examined.

The skin lotion with the aforementioned composition according to thepresent invention clearly demonstrated the dry skin amelioration effectcompared to the control. Further, no skin abnormalities such as rashesand dryness were observed.

Test Example 4 (Test of Measuring an HB-EGF Expression Rate)

The HB-EGF expression rates were investigated by the following testmethods where the compounds of the present invention were applied on thekeratinocytes.

1. Keratinocytes Used in the Test

Cultured keratinocytes of human epidermis origin, commercially availablefrom Kurabo Corporation, were used.

2. Measurement of HB-EGF Expression in the Keratinocytes

(1) Experimental Method

Keratinocyte susupension was adjusted to a concentration of 400×10⁴units/mL on an MCDB153 culture medium supplemented with BPE (bovinepituitary extract), insulin, ethanolamine, phosphoethanolamine, andhydrocortisone as growth promotors. Four milliliters of the cellsuspension was inoculated to a 60 mm plate (ex Falcon Corporation).Under an atmosphere of 95% (v/v) air and 5% (v/v) carbon dioxide gas, aconfluent state was achieved by static culturing at 37° C. for 3 days,while refreshing the culture media every other day.

After removing the culture medium supernatant by suction, the culturemedium was replaced with an MCDB 153 culture medium (containing insulin)to which dimethyl fumarate had been added at a final concentration of 50μmol/L or bis(diethylene glycol monoethyl ether) fumarate in 300 μmol/L.This plate was subjected to static culturing at 37° C. for 24 hoursunder an atmosphere consisting of 95% (v/v) air and 5% (v/v) carbondioxide gas. Using a QIAGEN™ RNA kit, whole mRNA was extracted. Eachamount of HB-EGF mRNA and G3PDH mRNA per total mRNA was determined byNorthern blotting. The amount of each mRNA per total in the absence ofthe fumarate was measured as the control. The amount of mRNA in thepresence of the fumarate was expressed in % relative to those of thecontrol.

(2) Results

The relative amounts of mRNA for HB-EGF and G3PDH in the presence ofeach sample are as shown below.

Amount of mRNA, % relative to the control Compounds added HB-EGF G3PDHBis (diethylene glycol monoethyl ether) 380% 98% fumarate Dimethylfumarate 470% 105% 

As can be seen from the above-mentioned results, bis(diethylene glycolmonoethyl ether) fumarate induced the expression of HB-EGF as stronglyas dimethyl fumarate which has sensitizing potential.

Test Example 5 (Test on Capability of Inhibiting Reduction in EpidermisThickness)

The following test method was used in order to investigate inhibition ofreduction in the epidermis thickness when the compound of the presentinvention was applied on aged skin.

1. Experimental Animals Used in the Test

Experimental animals used included 2 groups of aged hairless mice of 33weeks old at the beginning of the test with each 5 mice per group, andone group of 5 hairless mice of 9 weeks old at the beginning of thetest.

2. Measurement of the Epidermis Thickness

(1) Samples and the Experimental Method

A sample was prepared by adding bis(diethylene glycol monoethyl ether)fumarate to a 50% ethanol aqueous solution (base) at a concentration of0.5%. This sample was applied in an amount of 0.05 mL on the dorsal skinof hairless mice (in a diameter of approximately 2.5 cm) once a day forconsecutive 5 days per week followed by 2 days interval. This schedulewas repeated for 4 weeks. Subsequently, a portion of the skin wassampled and a frozen section was prepared. The thickness of theepidermis was measured by microscopic observation. The mean values ofthe epidermis thicknesses in hairless mice after the application of thefumarate are shown below.

Mean values of the Compound (Amount added) epidermis thickness, μm (33weeks old hairless mice) 20.96 Bis (diethylene glycol monoethyl ether)fumarate (0.5%) 17.24 Untreated (9 weeks old hairless mice) 28.88UntreatedThe results of this test indicates that thinning of the epidermis due toaging was clearly inhibited by the addition of bis(diethylene glycolmonoethyl ether) fumarate compared to the non-treatment.Test Example 6 (Test of Inhibition of the Expression of Amphiregulin)

The amount of expression of amphiregulin in the keratinocytes wasdetermined by the following test method.

1. Keratinocytes Used in the Test

Cultured keratinocytes of human epidermis origin (ex Kurabo Corporation)were used.

2. Measurement of Amphiregulin Expression in the Keratinocytes

(1) Experimental Method

Keratinocyte suspension was adjusted to a concentration of 150×10⁴units/mL in an MCDB153 culture medium supplemented with BPE (bovinepituitary extract), insulin, ethanolamine, phosphoethanolamine, andhydrocortisone as growth promotors. Four milliliters of the cellsuspension was inoculated to a 60 mm plate (ex Falcon Corporation).Under an atmosphere of 95% (v/v) air and 5% (v/v) carbon dioxide gas, aconfluent state was achieved by static culturing at 37° C. for 1 day.

After removing the culture medium supernatant by suction, the culturemedium was replaced with an MCDB 153 culture medium (containing insulin)to which bis(diethylene glycol monoethyl ether) fumarate had been addedin a final concentration of 300 μmol/L or dimethyl fumarate in 70μmol/L. This plate was subjected to static culturing at 37° C. for 24hours under an atmosphere consisting of 95% (v/v) air and 5% (v/v)carbon dioxide gas. Using a QIAGEN™ RNA kit, whole mRNA was extracted.Each amount of amphiregulin and G3PDH mRNA per total mRNA was determinedby Northern blotting. The amount of each mRNA per total in the absenceof the fumarate was measured as the control. The amount of mRNA in thepresence of the fumarate was expressed in % relative to those of thecontrol.(2) Results

The relative amounts of amphiregulin mRNA and G3PDH mRNA in the presenceof each sample are as shown below.

Amount of mRNA, % relative to the control Agents added AmphiregulinG3PDH Example 1. 8% 102% Bis (diethylene glycol monoethyl ether)fumarate Comparative Example 1 39%   98% Dimethyl fumarateThe fumaric acid diester derivative of the present invention was foundto inhibit the amphiregulin expression as strongly as dimethyl fumaratehaving sensitizing potential does. Since there was no difference in theamount of G3PDH mRNA between the control and Example 1, the mRNAexpression inhibition action by the fumarate diester derivative wasfound to be specific to amphiregulin.Test Example 7 (Test of Inhibition of Amphiregulin Expression when UsedTogether with Retinoic Acid)

The amount of amphireglurin expression in the presence of retinoic acidin the keratinocytes was determined by the following method.

1. Keratinocytes Used in the Present Test

Cultured keratinocytes of human epidermis origin (ex Kurabo Corporation)were used.

2. Measurement of Amphireglurin Expression in the Keratinocytes

(1) Experimental Method

Keratinocyte suspensions was adjusted to a concentration of 400×10⁴units/mL in an MCDB153 culture medium supplemented with BPE (bovinepituitary extract), insulin, ethanolamine, phosphoethanolamine, andhydrocortisone as growth promotors. Four milliliters of the cellsuspension was inoculated to a 60 mm plate (ex Falcon Corporation).Under an atmosphere of 95% (v/v) air and 5% (v/v) carbon dioxide gas, aconfluent state was achieved by static culturing at 37° C. for 3 days,while refreshing the culture every other day.

After removing the culture medium supernatant by suction, the culturemedium was replaced with an MCDB 153 culture medium (containing insulin)to which bis(diethylene glycol monoethyl ether) fumarate at a finalconcentration of 300 μmol/L or dimethyl fumarate at 70 μmol/L, andoptionally retinoic acid at 1 μmol/L had been added. This plate wassubjected to static culturing at 37° C. for 24 hours under an atmosphereconsisting of 95%(v/v) air and 5% (v/v) carbon dioxide gas. Using aQIAGEN™ RNA kit, whole mRNA was extracted. Each amount of amphiregulinmRNA and G3PDH mRNA per total mRNA was determined by Northern blotting.The amount of each mRNA per total in rhe absence of the fumarate wasmeasured as the control. The amount of mRNA in the presence of thefumarate was expressed in % relative to those of the control.(2) Results

The relative amounts of amphiregulin mRNA and G3PDH mRNA after theaddition of each sample are as shown below.

Amount of mRNA, % relative to the control Agents added AmphiregulinG3PDH Example 2.  70% 102% Bis (diethylene glycol monoethyl ether)fumarate + retinoic acid Comparative Example 2. 120%  98% Dimethylfumarate + retinoic acid Comparative Example 3. 300% 105% Retinoic acidaloneAs seen in Comparative Example 3, amphiregulin was strongly induced bythe addition of retinoic acid and the induced amphiregulin is consideredto be closely involved in formation of dry skin caused by retinoic acid.Example 2 reveales that the fumaric acid diester derivatives of thepresent invention more strongly inhibit the expression of amphiregulininduced by the addition of retinoic acid than dimethyl fumarate havingsensitizing potential does. Since the amount of mRNA for G3PDH as thecontrol in Example 2 agreed those in the Comparative Examples within anexperimental error, the function of the fumaric acid diester derivativeto inhibit the expression of mRNA was found to be specific toamphiregulin.Test Example 8 (Test of Inhibition of Dry Skin)

The effect of inhibiting dry skin was investigated by the following testmethod in which the present fumarate was applied to the dry skin inducedby retinoic acid.

1. Experimental Animals Used in the Test

Hairless mice of 10 weeks old at the beginning of the test were usedwith five mice per group.

2. Measurement of TEWL

A sample was prepared by adding dimethyl fumarate or bis(diethyleneglycol monoethyl ether) fumarate to a 50% ethanol aqueous solution(base) in a concentration of 0.5%. This sample was applied in an amountof 0.05 mL to the dorsal skin of hairless mice (in a diameter ofapproximately 2.5 cm) once a day for consecutive 5 days(pre-application). Subsequently, 0.05 mL of a 0.05% retinoic acidsolution in 50% ethanol aqueous solution was applied once a day for 4days. During these 4 days, retinoic acid was applied in the morning,and, some hours later in the afternoon, the sample was applied. The TEWLvalues were measured by a hydrograph AMU-100 (rx K & 5 Corp.) aftercompletion of the application. In Comparative Example 5, a controlsample comprising the base alone was used instead of the sample. InComparative Example 6, the base was always applied. The TEWL valuesafter the application of the samples are as shown below.

Mean value of TEWL, Group Components mg/cm²/min. Example 3 Bis(diethylene glycol 0.055 monoethyl ether) fumarate Comparative Example 4Dimethyl fumarate 0.063 Comparative Example 5 Retinoic acid, and no0.110 fumarate Comparative Example 6 Always the base only 0.012

As seen above, increase in TEWL due to the application of retinoic acidwas observed in Comparative Example 5 compared with Comparative Example6 where the base alone was applied. In Example 3, because of theaddition of the fumaric acid diester derivative of the presentinvention, apparently smaller increase in TEWL than in ComparativeExample 5 was observed, which indicates amelioration of dry skin inducedby retinoic acid. The TEWL value in Example 3 was almost equal to thatin Comparative Examle 4 where dimethyl fumarate having sensitizingpotential was used.

Test Example 9 (Test of Depression of UV-Induced EpidermalHyperproliferation)

The inhibition of epidermal hyperproliferation was investigated by thefollowing test method in which the present fumarate was applied to theskin where epidermal hyperproliferation had been induced by UVirradiation

1. Experimental Animals Used in this Test Example

Hairless mice of 10 weeks old at the beginning of the test were usedwith 5 mice per group.

2. Measurement of Epidermis Thickness

(1) Samples and Experimental Method

Samples were prepared by adding dimethyl fumarate or bis(diethyleneglycol monoethyl ether) fumarate to a 50% ethanol aqueous solution(base) at a concentration of 0.5%. Initially, this sample was applied inan amount of 0.05 mL to the dorsal skin of the hairless mice (in adiameter of approximately 2.5 cm) once a day and 5 times per week for 4weeks (pre-application). Subsequently on the 3^(rd) day from the lastapplication of the pre-application, the skin was irradiated with a UVBray once at a dose of 0.15 J/cm². The animals were slaughtered on the4^(th) day after the irradiation and skin section were made. From thesection, tissue section samples were prepared by HE staining. Thesamples prepared were photographed using an light microscope. In themicroscopic photographs, the thickness of the epidermis was measured.Using the epidermis thickness at the beginning of the test as astandard, the mean values were compared between the control group andthe treated groups.

The mean values of thickness of the epidermis after the application ofeach sample are as shown below.

Thickness of the Agents (Amount added) epidermis (μm) Bis (diethyleneglycol monoethyl 61.6 ether) fumarate (0.5%) Dimethyl fumarate (0.5%)40.4 50% ethanol aqueous solution 103.9 (base) No application 127.2The results of the test show that the addition of fumaric acid diesterderivatives inhibited epidermal hyperproliferation as shown in thereduced epidermal thickening by UVB, compared with the group treatedwith a base alone or the non-application group.Application Example 1 (Skin Cream)

A skin cream was prepared by mixing the fumaric acid diester derivativeaccording to the following composition.

(1) Composition

Mixing ratio Ingredients (mass %) (Component A) Beeswax 2.0 Stearic acid5.0 Stearyl alcohol 5.0 Hydrogenated lanolin 2.0 Squalene 20.0 Sorbitanmonostearate 3.0 Polyoxyethylene (20) 3.0 sorbitan monostearatePropylene glycol 5.0 (Component B) Methylparaben 0.2 Purified waterBalance to the total of 100 (Component C) Bis (diethylene glycol 10.0monoethyl ether) fumarate(2) Preparation Method

Bis(diethylene glycol monoethyl ether) fumarate as Component C was addedto component B. After dissolving Components A and B separately byheating them to 80° C., both components were combined and then cooled to30° C. while stirring to make a skin cream.

Application Example 2 (Skin Lotion)

A skin lotion was prepared by mixing the fumaric acid diester derivativeaccording to the following composition.

(1) Composition

Mixing ratio Ingredients (mass %) (Component A) olive oil 10.0 Isopropylmyristate 1.0 Polyoxyethylene (6) 0.5 nonylphenyl ether Propylene glycol1.0 Glycerin 2.0 (Component B) Methylparaben 0.1 Ethanol 7.0 Purifiedwater Balance to the total of 100 (Component C) Bis (diethylene glycol0.0001 monoethyl ether) fumarate(2) Preparation Method

Bis(diethylene glycol monoethyl ether) fumarate as Component C was addedto Component B. After dissolving Components A and B homogeneously,Components A and B were mixed and dispersed with stirring to prepare alotion. The lotion prepared was poured in a container. The content wasshaken to effect a uniform dispersion before use.

Application Example 3 (Bath Agent)

A bath agent with the following composition was prepared by a commonmethod.

Mixing ratio Ingredients (mass %) Bis (diethylene glyol 1.0 monoethylether) fumarate Sodium hydrogen carbonate Balance Sodium carbonate 20.0Sodium sulfate 15.0 Sodium chloride 7.5 Silica 0.5 1,3-butylene glycol1.0 Urea 1.0 Seaweed extract 1.0 Colorant Appropriate amount DextrinAppropriate amount Perfume Appropriate amountApplication Example 4 (Skin Cream)

A skin cream was prepared by mixing the fumaric acid diester derivativeand retinoic acid according to the following composition.

(1) Composition

Mixing ratio Ingredients (mass %) (Component A) Beeswax 2.0 Stearic acid5.0 Stearyl alcohol 5.0 Hydrogenated lanolin 2.0 Squalene 20.0 Sorbitanmonostearate 3.0 Polyoxyethylene (20) 3.0 sorbitan monostearatePropylene glycol 5.0 (Component B) Methylparaben 0.2 Purified waterBalance to the total of 100 (Component C) Bis (diethylene glycol 10.0monoethyl ether) fumarate Retinoic acid 1.0(2) Preparation Method

Bis(diethylene glycol monoethyl ether) fumarate and retinoic acid asComponent C were added to Component B. After dissolving components A andB separately by heating to 80° C., both components were combined andthen cooled to 30° C. with stirring to make a skin cream.

Application Example 5 (Skin Lotion)

A skin lotion was prepared by mixing the fumaric acid diester derivativeand retinoic acid according to the following composition.

(1) Composition of a Skin Lotion

Mixing ratio Ingredients (mass %) (Component A) Olive oil 10.0 Isopropylmyristate 1.0 Polyoxyethylene (6) 0.5 nonylphenyl ether Propylene glycol1.0 Glycerin 2.0 (Component B) Methylparaben 0.1 Ethanol 7.0 Purifiedwater Balance to the total of 100 (Component C) Bis (diethylene glycol0.0001 monoethyl ether) fumarate Retinoic acid 1.0(2) Preparation Method

Bis(diethylene glycol monoethyl ether) fumarate and retinoic acid asComponent C were added to Component B. After dissolving Components A andB homogeneously, Components A and B were mixed and dispersed withstirring to prepare a lotion. The lotion prepared was filled in acontainer. The contents were shaken to effect uniform dispersion beforeuse.

Application Example 6 (Skin Lotion)

A skin lotion was prepared by mixing the fumaric acid diester derivativeand retinoic acid according to the following composition

(1) Composition of a Skin Lotion

Mixing ratio Ingredients (mass %) (Component A) Olive oil 10.0 Retinylacetate 0.02 Isopropyl myristate 1.0 Polyoxyethylene (6) 0.5 nonylphenylether Propylene glycol 1.0 Glycerin 2.0 (Component B) Methylparaben 0.1Ethanol 7.0 Purified water Balance to the total of 100 (Component C) Bis(diethylene glycol 3.0 monoethyl ether) fumarate Retinoic acid 1.0(2) Preparation Method

Bis(diethylene glycol monoethyl ether) fumarate and retinoic acid asComponent C were added to Component B. After dissolving Components A andB homogeneously, Components A and B were mixed and dispersed withstirring to prepare a lotion. The lotion prepared was filled in acontainer. The contents were shaken to effect uniform dispersion beforeuse.

Application Examples 7 through 9 (Skin Cream)

Skin cream was prepared according to the following composition in aconventional method.

Mixing ratio (mass %) Application Application Application IngredientsExample 7 Example 8 Example 9 Bis (diethylene glycol 0.1 0.1 0.1monoethyl ether) fumarate Stearic acid 1 1 — Isostearic acid — — 1Glycerin monostearate 2 2 2 Behenyl alcohol 2 2 2 Bleached beeswax 1 1 —Cetyl myristate 1 1 1 Sorbitan sesquioleate 1 1 1N-stearoylphytosphyngosine 0.1 0.1 0.1 Hydrogenated lecithin 0.1 0.1 0.1Vegetable squalane 5 5 5 Retinyl palmitate 0.05 — — Octyldodecylmyristate 5 5 5 Phellodendron bark extract 0.1 1 0.1 Pyracanthafortuneana 0.1 0.3 — extarct Water-soluble glycyrrhiza — — 0.1 extract1,3-butylene glycol 5 10 5 Conc. Glycerin 5 5 5 Parahydroxybenzoateester 0.2 0.2 0.2 N-acetylglucosamine 0.1 0.1 0.1 oligomerMagnesiumascorbylphosphate 0.1 0.1 0.1 Sodium ascorbylphosphate 0.1 0.10.1 Gamma-aminobutyric acid 0.1 0.1 0.1 Sodium N-stearoylglutamate 0.20.2 0.2 Alkyl-modified carboxyvinyl 0.05 0.05 0.05 polymer*¹Nicotinamide 0.1 0.1 0.1 Sarcosine 0.1 0.1 0.1 Purified water BalanceBalance Balance *¹PEMULEN ™ TR-1, ex B F. Goodrich CompanyApplication Examples 10 and 11 (Lotion)

Lotions were prepared according to the following composition in aconventional method.

Mixing ratio (mass %) Application Application Ingredients Example 10Example 11 Bis (diethylene glycol 0.1 0.1 monoethyl ether) fumaratePhellodendron bark extract 0.1 0.3 Hibiscus extract 0.2 0.5 Lactic acidbacteria 0.1 0.1 fermentation solution 1,3-butylene glycol 5 5Dipropylene glycol — 5 Raffinose 1 1 Ethanol — 1 Phenoxyethanol 0.2 0.2Pectin — 0.05 Xanthan gum — 0.1 Sodium citrate 0.05 0.05 Horsetailextract 0.1 0.1 Diisopropylamine dichloroacetate 0.2 0.2Gamma-amino-beta-hydroxybutylic acid 0.2 0.2 Sodium hyaluronate 0.0010.001 Dipotassium glycyrrhizinate 0.2 0.2 Naematoloma sublateritumextract 0.05 0.05 Decarboxy carnosine hydrochloride 0.05 0.05 Perfume0.02 0.02 Purified water Balance BalanceApplication Examples 12 through 14 (Gel)

Gels were prepared according to the following composition in aconventional method.

Mixing ratio (mass %) Application Application Application IngredientsExample 12 Example 13 Example 14 Bis (diethylene glycol 0.1 0.1 0.1monoethyl ether) fumarate Decamethyl- 10 10 10 cyclopentasiloxaneIsostearyl isostearate 1 — — Olive oil — 1 — Macadamianut oil — — 1Eucalyptus oil 0.1 — 0.1 Hexyldecanol 1 0.1 — dl-alpha-tocopheryl — 0.1— nicotinate Retinol 0.1 — — Polyoxyethylene (60) 2 2 2 hydrogenatedcastor oil Spherical silicone 1 1 5 powder*² Phellodendron bark 0.1 10.1 extract Water-soluble chlorophyll 0.02 0.02 0.02 Salvia extract —0.3 0.1 1,3-butylene glycol 5 10 5 Sorbitol solution 3 3 3 Polyethyleneglycol 4000 1 1 1 Carboxyvinyl polymer 0.2 0.2 0.2 Glycoceramide*³ 0.10.1 0.1 Parahydroxybenzoate ester 0.2 0.2 0.2 Mevalonolactone 0.5 0.50.5 Edetic acid salt 0.02 0.02 0.02 Potassium hydroxide 0.05 0.05 0.05Purified water Balance Balance Balance *²Tospearl ™, ex GE ToshibaSilicone Corp. *³Bioceramide ™, ex Kibun Food Chemical Corp.Application Examples 15 through 17 (Lipophilic Cream)

Lipophilic creams were prepared according to the following compositionin a conventional method.

Mixing ratio (mass %) Application Application Application IngredientsExample 15 Example 16 Example 17 Bis (diethylene glycol 0.1 0.1 0.1monoethyl ether) fumarate Comodified silicons*⁴ 2 2 2 Polyoxyethylenemodified — 2 — silicone dispersion*⁵ Squalane — — 10Decamethylcyclopenta- 15 20 10 siloxane Retinol 0.05 0.05 0.05Methylpolysiloxane 5 2 3 Cholesteryl long-chain — — 3 branched fattyacids ester*⁶ Silicone elastomer 5 2 — dispersion*⁷ Phellodendron barkextract 1 1 1 Glycyrrhiza extract 0.1 0.1 0.1 Water-soluble chlorophyll0.02 0.02 0.02 Sodium chloride 1 1 1 Dipropylene glycol 5 5 5 Conc.Glycerin 5 5 5 Raffinose 1 1 1 Parahydroxybenzoate ester 0.3 0.3 0.3N-methyl-L-serine 0.5 0.5 0.5 Purified water Balance Balance Balanced*⁴ABIL ™ EM 90, ex Goldschmidt AG. *⁵Silicone BY22-008, ex Toray DowCorning Silicone Co. *⁶YOFCO CLE-NH, ex Nippon Seika Corporation Co.*⁷Trefil ™, ex Toray Dow Corning Silicone Co.Application Examples 18 through 20 (Sunscreen)

Sunscreens were prepared according to the following composition in aconventional method.

Mixing ratio (mass %) Application Application Application IngredientsExample 19 Example 20 Example 21 Bis (diethylene glycol 0.1 0.1 0.1monoethyl ether) fumarate Dioctyl ether 22 15 10 Comodified silicone*⁴ 22 2 Glyceryl — — 5 tri-2-ethylhexanoate Hydrogenated oil — — 0.1Methylphenyl polysiloxane — 3 — Macadamianut fatty acid — — 2phytosteryl ester 2-ethylhexyl — 7 7 4-methoxycinnamate Titanium oxide 5— 4 Zinc oxide 5 — 4 Phellodendron bark extract 1 1 1 Magnesium chloride1 1 1 1,3-butylene glycol 5 5 5 Phenoxyethanol 0.3 0.3 0.3 Hibiscusextract 1 1 1 Aloe extract 0.1 0.1 0.1 Yeast extract*⁸ 1 1 1 Purifiedwater Balance Balance Balance *⁴ABIL ™ EM90, ex Goldschmidt AG.*⁸DISMUTIN ™, ex Pentapharm AG.

INDUSTRIAL APPLICABILITY

The present invention provides an epidermal barrier function-enhancingagent and a composition for skin external application which are verysafe, quickly ameliorate the epidermal permeability barrier functiondisruption, and have excellent effects on maintaining the skin in acosmetologically healthy state; a HB-EGF expression-inducing agent toenhance epidermal turnover; an amphiregulin expression inhibitor todeminish epidermal hyperproliferation; an agent to be used together withretinoic acid or a derivative thereof to prevent dry skin caused byretinoic acid or a derivative thereof; and an ameliorating agent foraged skin which ameliorates metabolic disruption and skin thinning ofthe skin due to aging.

1. A skin external agent composition comprising fumaric acid diesterderivative expressed by the following general formula (1)

wherein R¹ represents a C₁₋₄ alkylene group; R2 and R3 each represents alinear or branched C₁₋₈ alkyl group; and n is an integer of 2 to 5, andretinoic acid or a derivative thereof, wherein the fumaric acid diesterderivative is contained in the amount of 0.001 to 15.0% and retinoicacid or a derivative thereof is contained in an amount of 0.001 to 1%relative to a total amount of the epidermal external agent composition.